1. Technical Field
The present invention generally pertains to the field of modulating activity of G protein-coupled receptors (GPCR) and of identifying and preparing G protein coupled receptor antagonist and agonist compounds, including direct, indirect, full, partial, inverse and allosteric agonists. The invention also encompasses compounds that bind to GPCR to stabilize a particular conformation of the GPCR. These compounds can serve as lead compounds for drug discovery purposes or for studying the GPCR three dimensional structure of specific conformations by such methods as X-ray crystallography or NMR. The invention also relates to an approach using high-throughput screening to identify small molecules that can bind to GPCRs and modulate their function by affecting the way in which they contact their cognate G protein(s). As a first step in identifying GPCR modulators, peptide analogs are identified that mimic or antagonize G proteins and bind with high affinity to the particular receptor under study. These peptides then are tested for their specificity. The most specific peptides are used in a competitive assay to screen for small molecules or other peptides that can, for example, (1) increase the binding of the high affinity peptide (“super agonist”) or (2) can decrease the binding of the high affinity peptide, presumably by competing for binding at the GPCR (“antagonists”).
2. Description of the Background Art
A great number of chemical messengers exert their effects on cells by binding to G protein-coupled receptors (GPCR). GPCR include a wide range of biologically active receptors such as hormone receptors, viral receptors, growth factor receptors, chemokine receptors, sensor receptors and neuroreceptors. These receptors are activated by the binding of ligand to an extracellular binding site on the GPCR and mediate their actions through the various G proteins. The molecular interactions that occur between the receptor and the G protein are fundamental to the transduction of environmental signals into specific cellular responses.
G protein-coupled receptors have seven transmembrane helices which form, on the intracellular side of the membrane, the G protein binding domain. Experiments have suggested that activation of the receptor by ligand binding changes conformation of the receptor, unmasking G protein binding sites on the intracellular face of the receptor. The transduction of the signal from the extracellular to intracellular environments requires the actions of heterotrimeric G proteins. The molecular interactions that occur between the receptor and the G protein are fundamental to the transduction of environmental signals into specific cellular responses. Heterotrimeric G proteins are thought to interact with GPCR in a multi-site fashion with the major site of contact being at the carboxyl terminus of the Gα subunit. Hamm et al., Science 241:832-835, 1998; Osawa and Weiss, J. Biol. Chem. 270:31052-31058, 1995; Garcia et al., EMBO J. 14:4460-4469, 1995; Sullivan et al., J. Biol. Chem. 269:21519-21525, 1994; West et al., J. Biol. Chem. 260:14428-14430, 1985.
In the inactive state, G proteins are heterotrimeric, consisting of one α, one β and one γ subunit and a bound deoxyguanosine diphosphate (GDP). Following ligand binding, the GPCR becomes activated. Conformational changes in the activated receptor lead to activation of the G protein, with subsequent decreased affinity of Gα for GDP, dissociation of the GDP and replacement with GTP. Once GTP is bound, Gα assumes its active conformation, dissociates from the receptor, and activates a downstream effector. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as both an intermediate that relays the signal from receptor to effector and as a clock that controls the duration of the signal. A variety of studies have implicated the carboxyl terminus of G protein α subunits in mediating receptor-G protein interaction and selectivity.
The carboxyl terminal 11 amino acids are most important to receptor interaction and to the specificity of this interaction. Martin et al., J. Biol. Chem. 271:361-366, 1996; Kostenis et al., Biochemistry 36:1487-1495, 1997. Other regions on Gα also are involved in receptor contact, however. Portions of the Gβγ dimer also have been implicated in GPCR binding. See Onrust et al., Science 275:381-384, 1997; Lichtarge et al., Proc. Natl. Acad. Sci. USA 93:7507-7611, 1996; Mazzoni and Hamm, J. Biol. Chem. 271:30034-30040, 1996; Bae et al., J. Biol. Chem. 272:32071-32077, 1997. The carboxyl terminal amino acid regions of Gα proteins (and other GPCR binding regions of the heterotrimeric G protein) not only provide the molecular basis of receptor-mediated activation of G proteins, but also play an important role in determining the fidelity of receptor activation. Conklin et al., Nature 363:274-276, 1993; Conklin et al., Mol. Pharmacol. 50:885-890, 1996.
The involvement of the carboxyl-terminal 11 amino acids of Gt (amino acids 340-350) in interactions with the activated GPCR (R*) is suggested by many studies, including (a) the finding that Pertussis toxin catalyzes the ADP-ribosylation of Cys0347, which uncouples Gt from R*; (b) a peptide corresponding to amino acids 340-350 of Gt can uncouple R* from Gt and can itself bind to R* and mimic the effects of Gt; (c) site-directed mutagenesis; and (d) the demonstration in related G proteins that specificity of coupling to particular receptors resides in their carboxyl terminus in interacting with R*.
The G proteins play important and intricate roles in determining the specificity and temporal characteristics of the cellular response to the ligand-binding signal. Hamm and Gilchrist, Curr. Opin. Cell Biol. 8:189-196, 1996. Multiple receptors can activate a single G protein subtype, and in some cases a single receptor can activate more than one G protein, thereby mediating multiple intracellular signals. In other cases, however, interaction of a receptor with a G protein is regulated in a highly selective manner such that only a particular heterotrimer is bound.
Recognition sites are the precise molecular regions on receptors to which the activating molecules bind. An agonist is an endogenous substance or a drug that can interact with a receptor and initiate a physiological response. A drug may interact at the same site as an endogenous agonist (i.e., hormone or neurotransmitter) or at a different site. Agonists that bind to an adjacent or a different site are termed allosteric agonists. As a consequence of the binding to allosteric binding sites, the interaction with the normal ligand may be either enhanced or reduced. The conformational change which the allosteric modulators induce in receptors concerns not only the binding domain for the classical ligands, but also the domain responsible for the interaction between the receptors and the G proteins.
The visual system is an example of one in which G protein signaling is important. Rod cells of the retina make up 95% of the photoreceptors and are highly sensitive to light. Rods allow vision at night or under conditions of very dim illumination. The rod visual protein rhodopsin resides in disk membranes in the rod outer segment (ROS). Rhodopsin is a prototypical GPCR. Helmreich and Hofmann, Biochim. Biophys. Acta 1286:285-322, 1996; Menon et al., Physiol. Rev. 81:1659, 2001; Teller et al., Biochemistry 40:7761, 2001. Rhodopsin is unique among GPCRs as it is not ligand activated.
Night vision relates to the ability of the organism to discriminate between slight differences in the intensity of dim light and, when dark-adapted, to detect small changes in light. Some persons report consistent difficulties in seeing at night, even when their eyes are fully dark-adapted. They cannot detect objects readily visible to others and show both confusion and slow recovery after brief exposure to relatively bright light sources. Maneuvering in dimly illuminated spaces and driving or flying at night present serious problems to these individuals. In addition, some individuals have nyctalopia, or true night blindness, which is diagnosed on the basis of a measurement of retinal sensitivity.
No definitive data on the occurrence of nyctalopia in the population are available, since measurements have never been made on a representative sample of the population. Studies of select groups (e.g., school children, service men), show that the normal population includes a percentage of persons of low visual sensitivity whose performance will be as poor as or poorer than that of many individuals whose nyctalopia is associated with disease or degenerative processes. For example, about 2 percent of Navy men were disqualified for night duties as “night blind” on this basis. It is also a disease of aging. As the general population ages, incidence of night blindness increases. Night blindness also has been observed in several diseases including:    (1) Retinitis pigmentosa (In the early stages of the disease, dark adaptation takes place, but at a retarded rate. As disease advances, rod function is progressively lost, and the absolute terminal threshold is elevated. More than 100,000 Americans have retinitis pigmentosa, and most people with retinitis pigmentosa are blind by the age of 40. See Farrar et al., EMBO J. 21(5):857-864, 2002;    (2) Glaucoma (Early impairment and progressive loss of rod sensitivity is observed in glaucoma. Cursiefen et al., Doc. Ophthalmol. 103(1):1-12,2001. Glaucoma is one of the leading causes of blindness in the U.S and one of the most common causes of blindness in individuals over age 60, one of the fastest growing groups in the U.S.);    (3) LASIK (Recent studies indicate a significant number of patients who undergo LASIK surgery fail a night vision test (30-60%). Miller et al., CLAO J. 27:84-88, 2001; Brunette et al., Ophthalmology 107:1790-1796, 2000;    (4) Side effects of drugs (Several medications can cause night blindness, including Methyltestosterone, Quinidinesis, Paramethadion and Trimethadione (anticonvulsants), Questran (cholesterol-lowering), Accutane (anti-acne), Hydroxychloroquine (anti-malarial), Videx (HIV), and Nefazodone (antidepressant)). Thus, the usefulness of a pharmaceutical approach to night blindness is clear. As the population ages, the number of affected individuals will increase.
Human dietary vitamin A deficiency can cause night blindness, and this can be reversed with vitamin A supplements. However, the night blindness associated with visual diseases such as retinitis pigmentosa (RP), cataracts, diabetic retinopathy, and glaucoma is only somewhat helped with vitamin A supplements, which do not change the course of the disease. Many of the mutations that cause retinal degeneration and visual loss are in genes that encode photoreceptor cascade proteins; others are in genes that encode photoreceptor structural proteins. Pang and Lam, Hum. Mutat. 19:189, 2002. Mutations in rhodopsin, PDEβ, or Gαt have been identified in different forms of congenital stationary night blindness. Pepe, Prog. Retin. Eye Res. 20:733-759, 2001. Stationary night blindness is not associated with retinal degeneration and manifests itself in the inability to see in the dark; daytime vision is largely unaffected. Congenital stationary night blindness (CSNB) refers to a group of non-progressive retinal disorders that are characterized predominantly by abnormal function of the rod system. Clinical heterogeneity even among family members with the same mutation raises the possibility that modifying factors, either genetic or environmental, influence the severity of the disease. Gottlob, Curr. Opin. Ophthalmol. 12:378-383, 2001.
In night blindness resulting from defects in rhodopsin, Gαt, or PDEβ, rod photoreceptors respond only to light intensities far brighter than normal, and the sensitivity of rods to light is similar to that of normal individuals who are not dark adapted. In fundus albipunctatus and in Oguchi disease, the rod photoreceptors can achieve normal sensitivity to dim light but only after 2 or more hours of dark adaptation, compared with approximately 0.5 hours for normal individuals. Dryja, Am. J. Ophthalmol. 130:547, 2000. In each of these forms of stationary night blindness, the poor rod sensitivity and the time course of dark adaptation correlate with the known or presumed physiologic abnormalities caused by the identified gene defects. Increasing the efficacy with which rhodopsin activates the phototransduction cascade is a possible new pharmacological approach to night blindness. Activated rhodopsin activates the rod visual G protein, Gt, which activates the visual transduction cascade. Pharmacologically increasing the effective signaling of rhodopsin can significantly impact people's ability to see and function in low light. The ability, therefore, to identify small molecule compounds that enhance the ability of G protein coupled receptors to signal would be a major benefit.
Because G proteins and their receptors influence a large number of intracellular signals mediated by a large number of different chemical ligands, considerable potential for modulation of disease pathology exists. Many medically significant biological processes are influenced by G protein signal transduction pathways and their downstream effector molecules. See Holler et al., Cell. Mol. Life Sci. 340:1012-1020, 1999. G protein-coupled receptors and their ligands are the target for many pharmaceutical products and are the focus of intense drug discovery efforts. Over the past 15 years, nearly 350 therapeutic agents targeting GPCRs have been successfully introduced into the market. Because of the ubiquitous nature of G protein-mediated signaling systems and their influence on a great number of pathologic states, it is highly desirable to find new methods of modulating these systems, including both agonist and antagonist effects. The ability to study the three-dimensional conformations of GPCRs in response to different individual ligands with different effects also is highly desirable, since these studies would aid in the search and development of drugs with particular structures which impart particular modulating effects on GPCRs.
Drug receptor theories are grounded in the law of mass action and include the concepts of affinity (the probability of the drug occupying a receptor at any given instant), intrinsic efficacy (intrinsic activity), which expresses the complex associations involving drug or ligand concentration, and activation states of receptors. Drugs classified as agonists interact with receptors to alter the proportion of activated receptors, thus modifying cellular activity. Conventional agonists increase the proportion of activated receptors; inverse agonists reduce it. Direct agonists act on receptors, while indirect agonists facilitate the actions of the endogenous agonist (the neurotransmitter itself). Allosteric modulation of receptor activation is a new approach which circumvents the development of tolerance.
Most currently available drugs affecting GPCRs act by antagonizing the binding between a G protein-coupled receptor and its extracellular ligand(s). On the other hand, receptor subtype-selective drugs have been difficult to obtain. An additional drawback to the classical approach of designing drugs to interfere with ligand binding has been that conventional antagonists are ineffective for some GPCRs such as proteinase activated receptors (PAR) due to the unique mechanism of enzymatic cleavage of the receptor and generation of a tethered ligand. In other cases, intrinsic or constitutive activity of receptors leads to pathology directly, thus rendering antagonism of ligand binding moot. For these reasons, alternative targets for blocking the consequences of GPCR activation and signaling are highly desirable. Increased understanding of the structural conformation of GPCRs under the influence of different agonists, antagonists or other ligands also allows design of compounds with highly specific effects on GPCRs.
One potential alternative target for inhibition by new pharmaceuticals has been the receptor-G protein interface on the interior of the plasma membrane. Konig et al., Proc. Natl. Acad. Sci. USA 86:6878-6882, 1989; Acharya et al., J. Biol. Chem. 272:6519-6524, 1997; Verrall et al., J. Biol. Chem. 272:6898-6902, 1997. The carboxyl terminus of Gα and other regions of the G protein heterotrimer conform to a binding site at the cytoplasmic face of the receptor. Sondek et al., Nature 379:311-319, 1996; Sondek et al., Nature 379:369-374, 1996; Wall et al., Science 269:1405-1412, 1996; Mixon et al., Science 270:954-960, 1995; Lambright et al., Nature 369:621-628, 1994; Lambright et al., Nature 379:311-319, 1996; Sondek et al., Nature 379:369-374, 1996; Wall et al., Science 269:1405-1412, 1996; Mixon et al., Science 270:954-960, 1995. Peptides corresponding to these binding regions or mimicking these regions can block receptor signaling or stabilize the active agonist-bound conformation of the receptor. Hamm et al., Science 241:832-835, 1988; Gilchrist et al., J. Biol. Chem. 273:14912-14919, 1998.
For example, in the case of rhodopsin, the rod photoreceptor, the Gα C-terminal peptide, Gα 340-350, stabilizes the receptor in its active metarhodopsin II conformation. Hamm et al., Science 241:832-835, 1988; Osawa and Weiss, J. Biol. Chem. 270:31052-31058, 1995. Two carboxyl terminal peptides from GαS (354-372 and 384-394), but not the corresponding peptides from Gαi2, evoke high affinity agonist binding to β2-adrenergic receptors and inhibit their ability to activate Gαs and adenylyl cyclase. Rasenick et al., J. Biol. Chem. 269:21519-21525, 1994. Thus, the carboxyl terminus of Gα is important in mediating the specificity of G protein responses. Drug discovery approaches which take advantage of this phenomenon, however, are not available. Jones et al., Expert Opin. Ther. Patents 9(12):1641, 1999.
In general, GPCRs require agonist binding for activation. However, for some receptors basic signaling activity may occur even in the absence of an agonist (constitutive activity). In addition, modifications to the receptor amino acid sequence can stabilize the active state conformation without the requirement for a ligand. Constitutive (agonist-independent) signaling activity has been demonstrated for both mutant and wild type (or native) form receptors (Tiberi and Caron, J. Biol. Chem. 269:27925-27931, 1994; Hasegawa et al., J. Biol. Chem. 271:1857-1860, 1996). A number of GPCRs that cause disease in humans, for example, receptors for thyroid-stimulating hormone (Vassart et al., Ann N. Y. Acad. Sci. 766:23-30, 1995), have been found to exhibit agonist-independent activity. An inverse agonist is an agent that binds to the receptor and suppresses this activity.
Experimentally, several single amino acid mutations have produced agonist-independent activity. β2 and α2 adrenergic receptors, for example, mutated at single sites in the third cytoplasmic loop, show constitutive activity. Ren et al., J. Biol. Chem. 268:16483-16487, 1993; Samama et al., Mol. Pharmacol. 45:390-394, 1994. In some cases, a large deletion mutation in the carboxyl tail or in the intracellular loops of GPCRs has led to constitutive activity. For example, in the thyrotropin releasing hormone receptor a truncation deletion of the carboxyl terminus or a smaller deletion in the second extracellular loop of the thrombin receptor renders the receptor constitutively active. Nussenzveig et al., J. Biol. Chem. 268:2389-2392, 1993; Matus-Leibovitch et al., J. Biol. Chem. 270:1041-1047, 1995; Nanevicz et al., J. Biol. Chem. 270:21619-21625, 1995.
These findings have led to a modification of traditional receptor theory. Samama et al., J. Biol. Chem. 268:4625-4636, 1993. It now is thought that receptors can exist in at least two conformations, an inactive conformation (R) and an activated conformation (R*), and that an equilibrium exists between these two states that markedly favors R over R* in the majority of receptors. It has been proposed that in some receptors (native and mutant) there is a shift in equilibrium in the absence of agonist that allows a sufficient number of receptors to be in the active R* state to initiate signaling. Therefore, in response to chemical or physical external stimuli, GPCRs undergo a conformational change leading to the activation of heterotrimeric G proteins which go on to initiate intracellular signaling events.
Several studies suggest that many GPCRs exhibit properties consistent with the existence of multiple conformational states. In rhodopsin, the existence of multiple conformers is evident from absorbance changes. Sakmar, Prog. Nucleic Acid Res. Mol. Biol. 59:1-34, 1998. Activation occurs by transition through intermediate conformations with the equilibrium between these forms showing a characteristic pH sensitivity. See Armis and Hoffman, Proc. Natl. Acad. Sci. USA 90:7849-7853, 1993; Vogel and Siebert, Biochemistry 41:3529-3535, 2002. Pharmacological studies suggest that the existence of distinct receptor conformers can have functional significance. Studies of fusion proteins of beta adrenergic receptor and G proteins suggest that partial agonists stabilize a conformational state distinct from that stabilized by a full agonist. Seifert et al., J. Pharmacol. Exp. Ther. 297:1218-1226, 2001.
The observation in several receptors that different agonists acting at the same receptor can direct the relative activation of downstream pathways, a phenomenon called “signal trafficking,” also suggests the presence of multiple populations of active receptor conformers. Kenakin, Trends Pharmacol. Sci. 16:232-238, 1995; Berg et al., Mol. Pharmacol. 54:94-104, 1998; Cordeaux et al., J. Biol. Chem. 276:28667-28675, 2001; Marie et al., J. Biol. Chem. 276:41100-41111, 2001. Fluorescence studies also suggest the presence of different receptor conformational populations when complexed with functionally distinct agonists. Ghanouni et al., J. Biol. Chem. 276:24433-24436, 2001. This emerging support for the existence of distinct, functionally relevant conformers in several GPCRs suggests that, for these receptors, the molecular activation mechanism must provide the means for switching among multiple conformations. A method to study these conformers by methods such as crystallographic methods and NMR would be highly useful in the process of discovering compounds which can modulate or stabilize particular conformers.
Protein-protein interactions involved in regulatory phenomena are reversible and tend to involve only a small fraction of the protein surface. Generally, to identify peptides that block the protein-protein interactions of interest particular peptides are synthesized in an attempt to mimic sections of one of the native interacting proteins or active sequences are selected from random peptide libraries after screening. Peptides are made up of sequences of amino acids, however unlike DNA recognition, which is linearly coded into the sequence, peptide binding is dependent on three-dimensional structure.
The visual pigment, rhodopsin, is the most extensively studied member of the family of G protein receptors. Recently, the X-ray structure of crystalline bovine rhodopsin has been determined to a resolution of 2.8 Å. This has paved the way for an understanding of the structure-function relationships of a prototypical GPCR at the molecular level. Since rhodopsin constitutes greater than 90% of the disk membrane protein, measurements made on the proteins of disk membranes predominantly reflect the properties of rhodopsin in its native environment. Rhodopsin consists of the apoprotein opsin and the chromophore 11-cis retinal. Opsin, consisting of 348 amino acids, has a molecular mass of about 40 kDa and folds into seven transmembrane helices of varying length and one short cytoplasmic helix. The retinylidene chromophore (the aldehyde of vitamin A1) is covalently bound to Lys-296 in helix 7 via a protonated Schiff base and keeps the receptor in an inactive conformation.
Light absorption causes a rapid 11-cis to all-trans isomerization of the chromophore which induces a series conformational of changes of the opsin moiety. This reaction occurs with high efficiency (quantum yield 0.67) and the primary photoproduct, photorhodopsin, is formed within a very short time (200 fs). Subsequently, photorhodopsin thermally relaxes within a few picoseconds to a distorted all-trans configuration, bathorhodopsin. On a nanosecond time scale, bathorhodopsin establishes an equilibrium with a blue-shifted intermediate before the mixture decays to form lumirhodopsin. Lumirhodopsin then is transformed into metarhodopsin I and subsequently metarhodopsin II, the active conformation for G protein coupling. Thus, there are two conformational switches in rhodopsin which are controlled by the protonation of specific amino acids of the protein: the transition from the inactive Meta I state to the active Meta II state and, in the absence of bound retinal, the transition from the inactive to the active state of opsin. According to current models, the receptor is kept in an inactive conformation by electrostatic interactions between charged groups in the protein, which are neutralized by the proton uptake involved in the transition to an active state conformation.
The active receptor species Meta II decays slowly within minutes, by hydrolysis of the Schiff base and dissociation of the receptor into the apoprotein opsin and retinal. Researchers have shown that opsin is in a pH-dependent conformational equilibrium between an active and an inactive state. During the decay of Meta II at neutral pH, most structural changes of Meta II formation are reverted and the decay product opsin eventually adopts an active conformation similar to that of Meta II.
Four distinct steps can be observed in the process of GPCR activation: (1) creation of the signal by a photon or by ligand binding; (2) transduction of the signal through the membrane; (3) interaction with the G protein; and (4) activation of the second messenger. Although the phases clearly differ in the kind of processes taking place, they are not discrete and independent. For example, allostery between ligand binding and G protein binding has been observed for several GPCRs, as well as cation-dependent allosteric regulation of agonist and antagonist binding. Wessling-Resnick and Johnson, J. Biol. Chem. 262:12444-12447, 1987; Hepler and Gilman, Trends Biol. Sci. 17:383-387, 1992; Nunnari et al., J. Biol. Chem. 262:12387-12392, 1987; Neve, Mol. Pharmacol., 39:570-578, 1991; Neve et al., Mol. Pharmacol. 39:733-739, 1991.
A number of cytoplasmic proteins interact exclusively with light-activated rhodopsin (R*). Because the crystal structure depicts the inactive form of rhodopsin as not interacting significantly with cytoplamic proteins, this structure can provide only indirect information about the R* state. In addition, two regions of the cytoplasmic surface domain of inactive rhodopsin structure (amino acid residues 236-239 and 328-333) have not been fully resolved by crystal structure analysis. Therefore, tools which can stabilize particular conformers would be useful for studying structure of GPCRs such as rhodopsin.
Negative antagonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this activity. Negative antagonists appear to act by constraining receptors in an inactive state. Samama et al., Mol. Pharmacol. 45:390-394, 1994. Although first described in other receptor systems, negative antagonism has been shown to occur with GPCRs such as opioid, β2-adrenergic, serotonin type 2C, bradykinin, and D1B dopamine receptors. Schutz and Freissmuth, J. Biol. Chem. 267:8200-8206, 1992; Costa and Herz, Proc. Natl. Acad. Sci. USA 86:7321-7325, 1989; Costa et al., Mol. Pharmacol. 41:549-560, 1992; Samama et al., Mol. Pharmacol. 45:390-394, 1994; Pei et al., Proc. Natl. Acad. Sci. USA 91:2699-2702, 1994; Chidiac et al., Mol. Pharmacol. 45:490-499, 1994; Barker et al., J. Biol. Chem. 269:11687-11690, 1994; Leeb-Lundberg et al., J. Biol. Chem. 269: 25970-25973, 1994; Tiberi and Caron, J. Biol. Chem. 269: 27925-27931, 1994.
That being stated, the concept of constitutively active receptors offer insights which explain pathophysiologic conditions. For example, a constitutively active receptor in a disease process such as hypertension may no longer be under the influence of the sympathetic nervous system. In hypertension, a constitutively active GPCR may be expressed in any number of areas including the brain, kidneys or peripheral blood vessels. A newly recognized class of drugs (negative antagonists or inverse agonists) which reduce undesirable constitutive activity can act as important new therapeutic agents. Thus, a technology for identifying negative antagonists (or understanding and stabilizing the conformational change in a GPCR that binding a negative antagonist compound causes) of both native and mutated GPCRs has important predictable as well as not yet realized pharmaceutical applications. Furthermore, because at least some constitutively active GPCRs are tumorigenic, the identification of negative antagonists for these GPCRs can lead to the development of anti-tumor and/or anti-cell proliferation drugs.
Mutagenesis studies of the carboxyl terminal region of Gαt have identified several specific amino acid residues in this binding region crucial for Gαt activation by rhodopsin. Martin et al., J. Biol. Chem. 271:361-6, 1996. Substitution of three to five carboxyl-terminal amino acids from Gαq with corresponding residues from Gαi allowed receptors which signal exclusively through Gαi subunits to activate the chimeric α subunits and stimulate the Gαq effector, phospholipase C β. Conklin et al., Nature 363:274-276, 1993; Conklin et al., Mol. Pharmacol. 50:885-890, 1996. All of these studies suggest that Gα carboxyl peptide sequences are responsible for the specificity of the signaling responses of the individual G proteins. There are 16 unique Gα subunits (Gαi1, Gαi2, Gαi3, GαO1, GαO2, GαZ, Gαt, Gαq, Gα11, Gα14, Gαs, Gα12, Gα13, Gα15/16, GαOLF and Gαgust) thought to mediate specific interaction with different GPCRs, several hundred of which have been cloned. Thus, peptides corresponding to G protein regions which bind the GPCR could be used as competitive inhibitors of receptor-G protein interactions. Hamm et al., Science 241-832-835, 1988; Gilchrist et al., J. Biol. Chem. 273:14912-14919, 1998. Drug discovery approaches which take advantage of this opportunity, however, are not available. Jones et al., Expert Opin. Ther. Patents 9(12):1641-1654, 1999.
Identification of potent lead compounds for use in modern high throughput screening assays and computerized design of new compounds using information about the desired three-dimensional conformation of receptor molecules, for example, are important aspects of the modern drug discovery process. One of the major challenges confronting those using these types of methods is the difficulty of identifying useful binding compounds from very large combinatorial libraries of potential candidate molecules. When literally hundreds of thousands of compounds are screened, characterizing the compounds which test positive for binding, for modulatory activity or for stabilization of a conformation (including false positives) is an expensive and time-consuming process. Hence, a method which can identify potent and useful lead compounds for high throughput screening and useful binding partners for three dimensional conformational studies and which reduce the number of false positives in the screening process would be very desirable.